Maize (Zea mays L.), also known as corn, is a major worldwide crop that has a number of practical uses. Maize is used as a food source for both humans and animals as well as a source of carbohydrates, oil, protein and fiber. Many products are produced or extracted from maize, such as corn syrup, adhesives, food thickeners, industrial and medical absorbants, ethanol, as well as many other products.
Maize can be bred by self-pollination or cross-pollination. Maize has separate male (called the tassel) and female (called the ear) inflorescences on the same plant. Maize is naturally pollinated when wind blows pollen from the tassels to the silks that protrude from the tops of the developing ears.
Most maize is produced from hybrid seed. The production of hybrid maize seed requires the elimination or inactivation of pollen produced by the female parent. Incomplete removal or inactivation of the pollen provides the potential for self-pollination. This inadvertently self-pollinated seed may be unintentionally harvested and packaged with hybrid seed. Several methods have been developed which can be used to control male fertility and thus prevent self-pollination. These methods include manual or mechanical emasculation (commonly referred to as detasseling), cytoplasmic male sterility, genetic male sterility, gametocides and the like.
Most frequently, hybrid maize seed is produced by manual or mechanical detasseling. This method works as follows. Typically, alternate strips of two inbred varieties of maize are planted in a field and the pollen-bearing tassels are removed from the inbred which is to be used as the female, prior to pollen being shed. As long as there is sufficient isolation from sources of foreign maize pollen, the ears of the detasseled inbred will be fertilized only from the male inbred, and the resulting seed is a hybrid.
One of the problems with detasseling is that it is laborious (and hence expensive) and is sometimes, unreliable. One alternative, to detasseling involves the use of cytoplasmic male sterile (CMS) inbreds. Cytoplasmic male sterility is governed by factors in the maternal cytoplasm that induce sterility. Cytoplasmic male sterility can be used for reproduction of the female by fertilization with fertile pollen (i.e., from a plant which is not cytoplasmically male sterile).
Another alternative to detasseling involves the use of genetic male sterility. Genetic male sterility is governed by nuclear factors that induce sterility and inhibit the normal development of the anthers and pollen. Genetic male sterility is inherited according to Mendelian principles in which the alleles for sterility are recessive (ms) to the alleles for pollen fertility (MS). There are many methods for conferring genetic male sterility. For example, U.S. Pat. Nos. 4,654,465 and 4,727,219 disclose the use of multiple mutant genes at separate locations within the genome to confer male sterility. U.S. Pat. Nos. 3,861,709 and 3,710,511 describe the use of chromosomal translocations for conferring genetic male sterility. Other methods involve delivering into a plant a gene encoding a cytotoxic substance associated with a male tissue specific promoter or an antisense system in which a gene critical to fertility is identified and an antisense to that gene inserted in the plant (see EP 329 308 and WO 90/08828).
Another alternative to detasseling is the application of certain chemicals referred to as gametocides, pollen suppressants or chemical hybridizing agents. These chemicals block or kill viable pollen formation and hence produce a transitory male sterility. However, there are a number of factors which affect the usefulness of these chemicals such as the expense of the chemicals, genotype specificity and the time of the application.
Homozygous inbred maize lines are required from the production of maize hybrids. Homozygous inbred lines can be produced via pedigree breeding, where two maize inbred lines, each of which possesses different sets of desirable characteristics, are crossed. Superior plants are selected for the traits of interest and then selfed for a number of generations in order for the line to become increasingly inbred. This process of continued selfing and selection is continued for five or more generations. The result of such breeding is the production of lines which are genetically homogenous or inbred. Typically, the development period for an inbred line using this method is a minimum of five years, if not longer.
One way for reaching homogeneity more quickly or completely when developing an inbred line is through the use of haploid plants. Haploids are plants which contain only one-half of the chromosome number present in somatic cells, which are cells other than haploid cells. There are a few maize plants which are known to generate haploids spontaneously. For example, plants are known which possess an indeterminate gametophyte (ig) gene which generate haploids. Additionally, a line known as ‘Stock 6’ (See, Birchler, J. A., “Practical Aspects of Haploid Production,” The Maize Handbook, Freeling and Walbot (eds). pp. 386–388 (1996)) possesses a propensity to generate haploids. Utilization of either ig or ‘Stock 6’ in a cross will result in the production of some haploid plants in the progeny.
Alternatively, haploid plants can be produced using techniques known in the art. One such technique is anther culture. Anther culture is a method by which large numbers of haploids can be produced directly from anthers in vitro. Generally, anther culture involves isolating immature anthers from plants and placing them onto a medium which induces the cells within the anther, which would normally be destined to become pollen grains, to begin dividing and form a cell culture from which the haploid plants can be regenerated. A number of techniques for carrying out anther culture are known in the art and are disclosed in J. M. Dunwell, “Anther and Ovary Culture”, In S W J Bright and M G K Jones, (eds.), Cereal Tissue and Cell Culture, Martinus Nijhoff Publisher, 1985, Dordrecht, pp. 1–44 and U.S. Pat. Nos. 5,306,864, 5,322,789 5,445,961, and 5,602,310. Another method which can be used to produce haploids is microspore culture. Microspore culture is similar to anther culture except that microspores are used instead of anthers to produce haploid plants (See, Coumans, et al., Plant Cell Reports 7:618–621 (1989); Pescitelli et al., Plant Cell Reports, 7:673–676 (1989)). The advantage of anther or microspore culture is that it makes it possible to test a larger number of new mutations and gene combinations and to select among those for desirable traits.
Haploids obtained either spontaneously or from anther or microspore culture, are sterile. To remedy this sterility, the chromosome number of the haploid can be doubled. Sometimes, the chromosome doubling can occur spontaneously. However, many times an agent must be used to effect the chromosome doubling. Agents which can be used to effect such chromosome doubling, include, but are not limited to, colchicine or a mitotic spindle inhibitor. The doubling of the chromosome number results in doubled haploid plants which are completely fertile and inbred. Because these doubled haploid plants breed true, it makes the selection process for desirable traits more efficient. Such doubled haploid plants possessing desirable traits or characteristics can be used in pedigree breeding to produce commercially valuable hybrids.
There exists a number of different methods for introducing a desired trait or characteristic into a targeted maize germplasm source, whether such maize germplasm source is an inbred or hybrid plant.
In a one method, backcrossing is used to introduce a desired trait or characteristic into a targeted maize germplasm source (called a recurrent parent) by crossing the recurrent parent with a donor plant (which is not the recurrent parent) which expresses certain traits of interest, such as, but not limited to, disease resistance, high yield potential, good stalk strength, reasonable drought tolerance, etc. While the donor plant is preferably an inbred, it can also be any plant variety which is cross-fertile with the recurrent parent. The progeny resulting from this crossing are then backcrossed to the recurrent parent, progeny possessing desirable traits identified, and the process repeated. The process of backcrossing to the recurrent parent and selecting for the desired traits is repeated for five or more generations. The progeny resulting from this process are homozygous for loci controlling the trait(s) being transferred but will be like the recurrent parent. The last backcross generation is then selfed in order to provide for pure breeding progeny for the gene(s) being transferred.
In a second method, apomixis can be used to introduce a desired trait or characteristic into a targeted maize germplasm source. Apomixis involves the production of hybrid seed without sexual reproduction. Apomixis is a natural method of reproduction in some plants and results in offspring that are genetically identical (i.e. clones) to the mother plant, thus allowing improved hybrids, to breed true. This would permit commercial producers and resource-poor farmers to replant seeds they produce, a strategy not practical with hybrid varieties available today for crops such as maize. U.S. Pat. No. 5,710,367, herein incorporated by reference, describes apomictic maize that have two unidentified genes which are believed to control apomictic development of the egg, as well as certain methods for making apomictic maize.
In a third method, genetic engineering can be used to introduce a desired trait or characteristic into a targeted maize germplasm source. Genetic engineering refers to the sophisticated, artificial techniques which are used to transfer genes from one organism to a recipient organism. In agriculture, genetic engineering is used to create new plant varieties containing genes from other organisms which provide the recipient plant with improved traits, such as, but not limited to, improved yield, color, height, tolerance to frost, insect or disease resistance. However, there has been a great deal of apprehension about the release of genetically engineered plants into the environment. In the United States, the U.S. Department of Agriculture, the Food and Drug Administration and the Environmental Protection Agency, all regulate the use of genetically engineered organisms, including genetically engineered plants.
One of the biggest concerns regarding genetically engineered or genetically modified (hereinafter “GM”) crops involves the possible contamination of non-GM crops by GM crops. Specifically, there is concern that transgenes contained in GM crops will travel, either through pollen or seed, to adjoining fields where non-GM crops are being cultivated and pollinate the non-GM crops. This so-called “pollen contamination” or “indiscriminate hybridization” can be costly to a farmer engaged in farming using non-GM crops. Crop isolation distances, crop rotational and management practices have been developed in an effort to alleviate the problem of “pollen contamination”. For example, in the U.S., it is recommended that an isolation distance of 203.1 meters (about 0.126 miles) be established between GM and non-GM maize fields for the purposes of seed production. The presence of natural barriers can be used to reduce this isolation distance.
Thereupon, it is readily apparent that cross-pollinating plants, such as maize, require a mechanism for preventing indiscriminate hybridization, especially from GM plants. Although differences in timing and isolation distance may contribute to reproductive isolation, physiological barriers often are sufficient to prevent crossing, especially among wind-pollinated species. One such possible mechanism for preventing such indiscriminate hybridization is cross-incompatibility. However, in contrast to self-incompatibility, cross-incompatibility (hereinafter referred to as “CI”) is poorly characterized both genetically and physiologically.
In domesticated maize, CI ranges in degree from creating a preference among pollen classes up to preventing seed set. Genes responsible for these effects are called gametophyte factors (hereinafter “Ga”) because the efficiency of pollen function is affected (see Nelson, O. E., The Maize Handbook, M. Freeling and V., Walbot, eds. Springer-Verlag (1993)). Ga factors conferring only a preference among pollen genotypes are cryptic, influencing the transmission of linked genes and the competitive ability of pollen in mixtures. Examples that involve recognition between corresponding alleles in pollen and silks are Ga2, Ga4, Ga8, and certain combinations involving Ga1. Incompatibility leading to failure of seed set occurs in conjunction with the strong allele of Gal, specifically when Ga1-s Gal-s plants are pollinated with ga1 gal, the cross used to isolate commercial popcorn from the pollen of other maize plants. As a system of isolation, Ga1-s is imperfect because some maize strains carry a gal -s or yet another allele, Gal-m, which permits them to cross to strains containing both ga1 and Ga1-s. In these strains, the barrier breaks down.
Thereupon, there is a need in the art for genetically and physiologically well-characterized cross-incompatibility systems in maize which prevent the indiscriminate hybridization of maize plants from unwanted pollen sources.